Noninvasive detection of colorectal cancer and other gastrointestinal pathology

ABSTRACT

A method for isolating viable, biologically substantially pure exfoliated fecal colonocytes at normal ambient temperature is described. Immunocoprocytes and inflammatory cells indicative of certain gastrointestinal conditions and a noninvasive method for detecting colorectal cancer are set forth. Composition of transport and suspension media for isolation of colonocytes are detailed.

This is a divisional of application Ser. No. 09/292,358 filed Apr. 15,1999 now U.S. Pat. No. 6,335,193.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is related to isolated colonocytes enabling earlynoninvasive detection of colorectal cancer and other gastrointestinaldiseases. More particularly, the present invention is related toisolated, biologically substantially pure and viable immunocoprocytesand nonepithelial cells of lymphoid origin obtained from a small fecalsample. The invention is further related to providing a transport mediumand a dispersion or suspension medium for isolating viable colonocytesfrom a fecal sample at normal ambient temperature and a method fordetecting colorectal and other gastrointestinal pathology employing theisolated colonocytes of the present invention. The isolated colonocytesalso allow the study and determination of other anomalous conditions,symptoms, disorders or pathological conditions.

2. Prior Art

A common gastrointestinal malignancy in humans is colorectal cancer. Ithas been estimated that colorectal cancer accounts for approximately 14%of all cancer-related deaths in men and women in the United States andits incidence continues to be high (Boring et al, CA Cancer J. Clin.1994; 44:7-26). Early detection is a critical factor in successfultreatment of this cancer, as it is in the treatment of othermalignancies.

Screening approaches to detection of colon and colorectal tumors arepresently based on the use of (a) fecal occult blood test (FOBT), (b)flexible sigmoidoscopy, (c) double contrast barium enema, and (d)colonoscopy. Among these screening tests only FOBT, which is based on arelatively high probability of bleeding from colorectal tumors, isnoninvasive, simple and relatively inexpensive. However, frequent falsepositive and false negative results of the FOBT considerably limit itsspecificity and sensitivity. Other procedures are expensive andinvasive. Hence, there is a clear need for providing a simple,noninvasive, reliable and inexpensive method for detecting colorectalcancer, gastrointestinal (GI) tract diseases and other pathologicalconditions.

Colonocytes represent an important source of informational markermolecules that provide a picture of the immediate past metabolic historyof the GI tract of a subject. In addition, such cells are representativeof the cell population from a statistically large sampling framereflecting the state of the colonic mucosa along the entire length ofthe colon in a non-invasive manner, in contrast to a limited sampling bycolonic biopsy using an invasive procedure involving endoscopy.

Colonocytes undergo certain changes or transformations and carry certainbiological or chemical markers indicative of colonic pathologiesincluding precancerous and cancerous conditions. Therefore, thecolonocytes could serve as a valuable early indicator of the onset ofneoplastic processes and other pathophysiological changes in the GItract. Subtle changes in the genes and surface proteins are examples ofsuch neoplastic markers. In particular, Ki67, the cell surfaceglycoprotein CD44 and tumor-associated antigens 19-9 and lectin bindingare specific biomarkers of neoplastic transformation in the GI tract.There is also a strong correlation between the amount of DNA in isolatedcolonic cells and the presence of tumors, because rapidly dividing cellscontain more DNA. It is also well recognized that the development ofcolonic adenomatous polyps and cancer is a multistep process involvingactivation of oncogenes (ki-ras), inactivation of tumor-suppressor genes(p53 and APC), and alterations in the DNA mismatch repair genes.

Heretofore, it was generally understood in the art to which thisinvention belongs that exfoliated colonic cells are destroyed once theyare shed into the stools, the reason being that these cells startbreaking down as soon as they are exposed to the atmosphere. Theenzymes, mucus and the bacteria contained in the stool contribute to theprocess of destroying the colonic cells. Chilling the freshly collectedstool sample to the temperature below −20° C. has been described, forexample, in U.S. Pat. Nos. 5,094,956, 5,380,647 and 5,455,160 only forpreserving the chemical constituents, without regard to the cellularcomponents. Thus, these procedures do not retain cellular integrity andare not applicable for isolating intact, viable cells free of otherimpurities.

Dutta and Nair (Gastroenterology, 114:1333-1335, 1998) refer to Albaughet al (Int. J. Cancer, 52:347-350, 1992) and Iyengar et al (FASEB J. 5:2856-2859, 1991) for accomplishing isolation of viable colonic cells.Albaugh et al described a transport medium and a procedure to obtaincolonocytes from a stool sample based on earlier work of Iyengar et al.However, the transport medium of the prior art was different from thetransport medium of the present invention in as much as Albaugh et al'smedium consisted of a saline solution to which antibiotics were added inaddition to fatty acid free BSA. In other words, the prior art transportmedium was deficient at least in one criterion, i.e., in not having amucolytic agent which is an absolute necessity for the formulation ofthe transport medium in accordance with the present invention.

Furthermore, in Albaugh et al's system the stool sample after collectionhad to be kept cooled in ice while being trasported to the laboratoryfor further processing and could be preserved in ice only for about anhour. In contrast, the system of the present invention does not requirecooling in ice and achieves desirable results at the normal ambienttemperature which is an important feature of the present invention.Moreover, Albaugh et al state that although their cells had a viabilityin excess of 80%, the presence of phagocytes and other cells could notbe ruled out. Indeed, FIGS. 3 and 4 of Iyengar et al clearly show thatthe cellular preparations obtained were considerably impure. Thus, theprocedures of Iyengar et al and Albaugh et al may provide viablecolonocytes, but they are inadequate for the purpose of obtainingsubstantially pure colonocytes. In summary, heretofore it has not beenpossible to obtain a viable, biologically subtantially pure sample of aparticular cell type isolated at normal ambient atmospheric conditionsfrom a small fecal mass.

SUMMARY OF INVENTION

It is, therefore, an object of the present invention to provide viable,biologically substantially pure exfoliated fecal colonocytes isolated atnormal ambient temperature.

It is a further object of the present invention to provide viable,isolated, immunocoprocytes.

It is an additional object of the present invention to provide viable,isolated, inflammatory cells of lymphoid or non-lymphoid lineage.

It is a further object of the present invention to provide a transportmedium and a dispersion or suspension medium for isolating viableexfoliated fecal colonocytes at normal ambient temperature.

A further object of the present invention is to provide a noninvasivemethod for detecting gastrointestinal disorders including colorectalcancer, employing the exfoliated fecal colonocytes isolated at normalambient temperature in accordance with the teachings of the presentinvention.

Various other objects and advantages of the present invention willbecome evident from the detailed description of the invention and fromthe brief description of the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, aspects and advantages will be betterunderstood with reference to the drawings in which:

FIG. 1 is a schematic presentation of the process of isolating viableexfoliated colonocytes at normal ambient temperature from a small sampleof fecal material;

FIG. 2 presents histogram data from flow cytometry of isolatedcolonocytes in accordance with the present invention showing a puritygreater than 96%; and

FIG. 3 is a diagrammatic representation of classes of colonic cellsidentified based on their immunoglobulin characteristics.

DETAILED DESCRIPTION OF THE INVENTION

Various objects and advantages of the present invention are achieved byobtaining viable, homogeneous colonocytes of desired cell types isolatedfrom a fecal sample at normal atmospheric conditions and ambienttemperature.

It should be understood that unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Although any methods and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresent invention, the methods and materials described herein arepreferred. Unless mentioned otherwise, the techniques employed orcontemplated herein are standard methodologies well known to one ofordinary skill in the art. The materials, methods and examples are onlyexemplary and not limiting.

The term “substantially pure” as used herein means that the product ishomogeneous or uniformly of a single type with greater than 96% purity,usually 98%-99% pure, as determined by flow cytometry or by theprocedures described herein and is without material interference orcontamination of other types of cells.

Heretofore, the problem encountered with exfoliated colonocytes in fecalmatter was that these cells got destroyed as soon as exposed to thenormal atmospheric conditions due to the presence in the fecal matter ofproteolytic enzymes, microflora, mucus and the like. Hence, a system hadto be devised to inhibit the action of all those factors or elementswhich prevented the isolation of intact, living colonocytes at normalambient temperature from fecal matter. This is achieved by the presentinvention by formulating two different media: (1) a transport medium,and (2) a dispersion or suspension medium. The transport medium is madeof physiological saline solution containing an enzyme inactivatingamount of an enzyme trapping or protease sequestering agent, asufficient amount of a bacteriocidal agent to inhibit bacterialactivity, and a mucolytic amount of an agent that destroys mucuscontained in fecal matter.

Various enzyme trapping, bacteriostatic and mucolytic agents will besuggested to one of ordinary skill in the art and any suitable agentsthat do not interfere with the objects of the present invention could beused in the formulation of the transport medium in accordance with theteachings of the present invention.

Preferred among enzyme trapping agents are proteolytic activityinhibitors and animal proteins. Examples of suitable proteolyticactivity inhibitors include PMSF, pepstatin A, bestatin and chymostatin.The reagents that inhibit or deactivate enzymatic activity includeformaldehyde, metal chelating agents, heavy metal ions, certain aminoacids such as tyrosine and phenylalanine, and high concentrations ofzinc or inorganic phosphates. Suitable among animal proteins are thosethat are non-immune, water soluble compounds including serum albumins ofrabbit (RSA), goat (GSA), sheep (SHA), horse (ESA), bovine (BSA) andhuman (HSA) origin. Certain polyamino acids that do not interefere witha later assay procedure could also be used as enzyme deactivatingagents. Suitable examples of such polyamino acids are poly-L-lysine,poly-L-proline, poly-l-tyrosine and the like.

Suitable examples of bacteriostatic agents are sodium azide, sodiumbenzoate, antibiotics (such as penicillin, streptomycin, amphotericin B.gentamicin, polymyxin B and the Like). A preferred bacteriocidal agentis Thimerosal (Sigma Chemical Co.)

Suitable examples of mucolytic agents are guaifenesin, guaiacol,potassium iodide, β-mercaptoethanol, dithiothreitol, capsaicin,glycyrrhizin and the like. A preferred mucolytic agent is N-AcetylCysteine (Sigma Chemical Co.).

Puck's Saline G is a preferred source of physiological saline solution.

A preferred transport medium is prepared as follows:

Puck's Saline G 500 ml Sodium Bicarbonate 350 to 500 mgs BSA 2.5 to 15gms N-Acetyl Cysteine 250 to 500 mgs Thimerosal 100 to 300 mgs

The dispersion or suspension medium differs from the transport mediumwith respect to bacteriocidal agent, e.g., Thimerosal, which is omittedwhen preparing the dispersion or suspension medium.

Procedure for Isolating Substantially Pure Colonocytes at Normal AmbientTemperature:

Referring now to FIG. 1, a small stool sample 10 (about 1 to 5 gm) isplaced into a tube 11 containing a transport medium 12, after which thetube is closed with a stopper. The stool sample 10 is then thoroughlydispersed in medium 12 after which the contents of tube 11 are emptiedinto a double walled plastic bag 13 (e.g., Stomacher bag with a meshliner, Tekmar Co.) and diluted with the dispersion medium 14 (about 50ml/gm of stool, wet weight) and mixed in a mechanical mixer 15 (e.g.,Stomacher, Tekmar Co.) for about 30 seconds. The contents of the plasticbag are then allowed to settle down briefly. The aliqot 16 of theplastic bag is then pipetted into 50 ml polypropylene conical centrifugetubes 17 and underlaid with a heavy medium 18 having a density in therange of about 1.033 to 1.120 (preferably Histopaque 1119, SigmaChemical Co.) and centrifuged at about 200×g for about 30 minutes in atable top centrifuge with the brakes off.

The colonic cells accumulate as a band at the interface between theheavy medium 18 and the lighter suspension 16 above. This band 19comprising the desired colonic cells is removed by sucking out with aplastic transfer pipette 20 and placed in a new 50 ml centrifuge tube 21and diluted with about 40 ml of the dispersion medium 22. The suspensionof cells thus obtained is then centrifuged at about 900×g for about 10minutes after which the clear supernatant is discarded and the cellpellet 23 remaining at the bottom of the centrifuge tube is resuspendedin phosphate buffered saline (PBS) 24 containing about 1% bovine serumalbumin (BSA). The suspension is then again centrifuged at 900×g afterwhich the clear supernatant is discarded and the pellet 25, comprisingsubstantially biologically pure isolated exfoliated viable colonocytes,is recovered.

For determining the viability of the isolated colonocytes, a portion ofthe pellet 25 is dispersed in PBS/BSA medium 26 (1 ml/gm of stoolsample). Then, a {fraction (1/10)} dilution of the suspension is countedin a hemocytometer in the presence of trypan blue. As is well known to askilled artisan, the cells that do not take up trypan blue areconsidered to be viable and counted to determine the cell yield.

The tube or vial 11 may be made of any suitable material includingplastics, polystyrene, polypropylene and the like.

It is important to note that an inventive aspect of the presentinvention is the formulation of a transport medium and a dispersion orsuspension medium which together allow the exfoliated colonocytes foundin the fecal sample to remain viable at the normal ambient temperatureduring and after the isolation procedure. In other words, the inventionenables isolation of intact, living exfoliated colonocytes from a smallsample (e.g. 3 gm) of fecal material without chilling or freezing, atnormal ambient temperature ranging from about 22° C. to about 24° C.during the entire isolation process. About 8 to 10 million livingcolonic cells can be obtained from one gram of the stool sample inaccordance with the techniques of the present invention.

When maintained at the normal ambient temperature in the suspensionmedium of the present invention, the isolated colonocytes can be viablypreserved for several hours. Table 1 shows cell yields and viability asa function of storage time and temperature conditions.

Of course, alternate techniques could be substituted in the isolationsteps. For example, the suspension of the stool sample in the transportmedium 12 may be filtered through screens (149 micrometers, 105micrometers and 52 micrometers). Also, the pellet in the dispersionmedium can be gently overlayered on higher density Percoll gradients andcentrifuged so that the cells can be recovered from the top of thegradient. Such modifications are common in the art and are includedwithin the purview of this invention.

It is understood, of course, that whenever appropriate a reference orbase line would be usually established using colonocytes obtained fromdisease-free subjects so that a comparative, diagnostic or evaluationstudy could be made with colonocytes obtained from a subject suspectedof a disease or pathological condition.

It was discovered that an important and advantageous feature of thenon-invasively obtained colonocytes of the present invention is thatthese isolated colonic cells carry markers or transformationscharacteristic of the pathology of the GI tract and, therefore, they canserve as diagnostic and predictor indicators of the GI tract pathology.

Immunocoprocytes

Since the colonic cells isolated from stools were discovered to be truerepresentative of the anatomical and pathophysiological condition of theentire colon, among other utilities these cells also allow monitoring ofmucosal immunity. The mucosa of the GI tract is a major site for theelaboration of immunological defenses mediated by immunoglobulins. Itwas discovered that a functionally distinctive group of cells,designated herein as immunocoprocytes, can be identified and isolatedfrom the exfoliated cells obtained by the methodologies of the presentinvention. Immunocoprocytes are unique in expressing a specificimmunoglobulin designated herein as IgC which is defined as a chimericimmunoglobulin that is recognized by antibodies both to IgG and IgA.Furthermore, immunocoprocytes are clonal, antigen-specific andcharacterized by the presence of Fc receptors and immunoglobulin A (IgA).

Given the affinity of immunocoprocytes to IgG and IgA antibodies,several approaches to isolate the immunocoprocytes would be suggested toone of ordinary skill in the art. For example, selective isolation ofimmunocoprocytes from a mixture of cells obtained from stools, colonicpurges or washings, or from surgical and autopsy specimens can beachieved using anti-IgG or a specific anti-IgC monoclonal antibody. Theindirect immune adherence approach utilizes a panning technique to allowthese cells to adhere to petri dishes coated with anti-IgG or specificanti-IgC antibodies. The use of anti-IgG antibody as a capture agent forimmunocoprocytes is based on the discovery in accordance with thepresent invention that pure IgG expressing mono-specific (viz., lackingco-expression of IgA) colonic cells are not detected under normalconditions. Preparation of desired types of monoclonal antibodies arewell known to one of ordinary skill in the art and are routinelyobtained.

Another approach to obtaining substantially pure immunocoprocytes is theuse of fluorescence-activated cell sorting (FACS) technique employingfluorochrome-conjugated anti-IgG or Anti-IgC. In one embodiment, coloniccells are incubated with FITC (fluorescein isothiocyanate) labelled IgG,the excess reagent is washed off and the fluorescently-taggedimmunocoprocytes are sorted in a fluorescence-activated cell sorter.

In another embodiment, monoclonal antibodies to IgG or IgC is covalentlyor non-covalently attached to a solid matrix, such as agarose beads,glass beads, polystyrene beads, hollow fiber, magnetic beads, plastictissue culture dishes and the like well known to a skilled artisan.Cells that adhere to the antibody coated support are separated from thecell suspension by simply separating the matrix from the suspension bymechanical, magnetic or any other suitable means. As it would be knownto one of ordinary skill in the art, the immunocoprocytes can also bebound to the surface of tissue culture flasks coated with anti-IgG oranti-IgC through a linker and after incubating the colonic cellsuspension in the flasks, unbound non-immunocoprocytes are decanted off.Bound immunocoprocytes are then recovered by scraping or by appropriateenzymatic cleavage of the linker. Linkers bound to bead matrices (e.g.,sepharose) are commercially available (e.g. Pharmacia).

Two-color immunofluorescent flow cytometry is used to determine thenumber or population of the immunocoprocytes. Colonocytes are incubatedwith anti-IgG FITC (green fluorescence) and anti-IgA PE (phycoerythrin,red fluorescence). After washing the cells with a buffer to remove theexcess antibodies, the cells are then analyzed in a flow cytometer tocount the cells with single fluorescence (green or red) and those cellswith double fluorescence (both green and red). Cells with doublefluorescence are the chimeric Ig C immunocoprocytes, while cells withthe red fluorescence are IgA secreting colonic epithelial cells. In mostcolonic preparations from normal subjects, there is no measurable numberof cells recognizing only anti-IgG FITC. In other words, colonocytesbearing only IgG are rare and may be associated with abnormal mucosal orsystemic immune dysfunction when they are present.

Analyses of immunofluorescently labelled cells by flow cytometry haverevealed the existence of at least three types of colonocytes. Todistinguish the Fc receptor of immunocoprocytes from other Fc receptors,the Fc receptor of immunocoprocytes has been designated herein as CFcreceptor. Table 2 shows some representative normal distribution of IgC,CFc and IgA found in these cells. A deviation from the normal valueswould indicate a disease process involving the immune system. FIG. 3 isa diagrammatic representation of at least three types of colonocytesgenerally detected: “A” shows immunocoprocytes with several uniquechimeric immunoglobulin IgC represented by two antibody binding sites oneach molecule 2 and several CFc receptors 3. “B” shows colonocytessimilar to “A”, but with several IgA immunoglobulin molecules 5 alongwith several CFc receptors 3. “C” shows colonocytes similar to “A” and“B”, but with no immunoglobulin and only CFc receptors.

Immunocoprocytes, IgA bearing colonocytes and CFC receptor bearingcolonocytes described above have distinct roles in immune surveillanceof the GI tract and in maintaining systemic humoral immunity of thetotal organism. These cells perform vital functions: (i) maintaining abalance in the colonization of the colon by microflora; (ii) they areclonal and contain a population of pluripotent cells each onerecognizing a single antigen, soluble or particulate, of dietary orbiological origin; (iii) they may act as antigen presenting cells togut-associated lymphoid tissue; (iv) they can be sentinels for detectinginvasion by pathogens (e.g., rotavirus, shigella, polio, intestinalparasites, mycobacteria and the like); and (v) their absence can signifya state of immunologic anergy of iatrogenic origin or congenitalimmunoglobulin deficiencies. Because these colonocytes including theimmunocoprocytes, are antigen-specific, they can be immortalized bytransformation with carcinogens, oncogene DNA, EBV, SV-40 and the like,to produce antibody-secreting cell lines specific for the selectedantigen by hybridoma technology well known to one of ordinary skill inthe art.

The following examples illustrate specific utilities of the identifiedor isolated colonocytes of the present invention. These examples areonly illustrative and not limiting in any manner.

EXAMPLE-1

Colonic cells isolated from normal subjects by the procedures describedin the present invention are substantially free of any inflammatorycells. In inflammatory bowel disease (IBD), such as ulcerative colitisand Crohn's disease, a significant number of inflammatory cells aremobilized to the surface of the colonic mucosa and are exfoliated alongwith cells of the epithelium.

One to two gram aliquots of stool collected from IBD patients, suspendedin the transport medium are homogenized in a Stomacher with about 150 mlof suspension medium. Aliquots (30 ml) of the suspension are underlaidwith 10 ml of Histopaque 1077 (density of 1.077) and centrifuged at roomtemperature at 200×g for 30 minutes with the brakes off. The interfacebetween the aqueous suspension and the Histopaque 1077 is recovered andwashed three times by repeated centrifugation. In this process,inflammatory cells are recovered in addition to the normal complement ofcolonic cells.

The inflammatory cells in the mixture of cells are tagged withanti-CD45/FITC (green fluorescence) and the positive cells are countedin a flow cytometer. CD45 (cluster of differentiation) also known asleucocyte common antigen, is a lineage-specific marker for lymphoidcells and are present on inflammatory cells. In addition a second markerof inflammation, anti-COX-2/PE (red fluorescence) may also be employedto detect inflammatory cells. Since colonic epithelial cells arenegative for CD45, the number of CD45-positive cells in an isolate is adirect measure of the severity of the inflammatory process in IBD.Monitoring of cells positive for both CD45 and COX-2 is an extremelyuseful non-invasive procedure for following the progression of thedisease during the course of treatment.

EXAMPLE-2

Assessment of Status of Mucosal Immunity:

Cells are obtained as described above from subjects who are suspected ofhaving an immunocompromised gut. Aliquots of cells (about 110K) aresuspended in PBS buffer and incubated at 37° C. for about 45 minuteswith one of the following combinations of fluorescently labelledantibodies: anti-IgG FITC (green), anti-IgA PE (red), and anti-IgCFITC+anti-IgA PE. A parallel tube containing cells with an isotypecontrol antibody is also maintained to account for non-specific bindingantibody. Direct immunofluorescence assays are conducted to measure thebinding of antibodies to different sets of colonic cells. A significantdecrease in number of immunocoprocytes (expressing IgC) or IgA bearingcells is of diagnostic significance for immune deficiency. Table 2 listsvalues obtained for normal subjects. Any deviation from the normalvalues wound be indicative of immune dysfunction. It should be notedthat direct and indirect immunofluorescence assays can be similarlycarried out for the assessment of a repertoire of macromolecules, suchas cytokines, signal transduction intermediates, growth factors and thelike.

EXAMPLE-3

Expression of Colon Cancer-Associated Biomarkers:

Cells are obtained as described above from patients suspected of havingcolon cancer or precursors of colon cancer (polyps). In one embodimentof this technique, cells are subjected to indirect immunofluorescenceassay for the expression of CD44 or its molecular variants, e.g.,CD44V3, CD44V6 and CD44V10; the presence of CD44 or its molecularvariants being diagnostic of colon cancer.

EXAMPLE-4

As a source of somatic cells obtainable non-invasively, the colonocytesof the present invention are representative of the phenotype as well asgenotype. Thus, they are useful in DNA typing and examination ofbiological macromolecules (such as DNA, RNA, protein and the like) fordetermining responses, for example, to pharmacologic and environmentalagents and assessment of multi-drug resistance. These isolated cells arealso useful in various other ways easily suggested to a skilled artisan.

It should be apparent that given the guidance, illustrations andexamples provided herein, various alternate embodiments, modificationsor manipulations of the present invention would be suggested to askilled artisan and these are included within the spirit and purview ofthis application and scope of the appended claims.

TABLE 1 Effect of storage on cell yields and viability Storage Time CellYield Viability (Hrs) Conditions % % 1-6 Ambient 100  85+ 10 Ambient 10065 24 4° C. 40 50 36 4° C. 24 5-6

TABLE 2 Distribution of IgC, IgA and CFc bearing colonic cells SubjectCode IgC CFc Receptor IgA 308-S1 18.5 90.3 46.5 308-S2 20.8 87.8 42.0318-S1 12.2 86.9 47.7 318-S2 26.5 91.2 37.7 319-S1 27.5 88.9 44.0 319-S232.9 90.9 30.0 325-S1 20.2 88.9 40.0 325-S2 16.8 82.2 24.0 Note: Thenumbers represent the % of total cells that carry the correspondingmolecule. These results were obtained by flow cytometric analysis ofimmunofluorescently labelled colonic cells isolated by the technologydescribed in this application.

Having thus described our invention, what we claim as new and desire tosecure by Letters Patent is as follows:
 1. A method for diagnosinginflammatory bowel disease, comprising the step of noninvasivelydetermining the presence of inflammatory cells in a population of cellsisolated from a stool sample of a subject suspected of inflammatorybowel disease, wherein the isolation of cells from a stool samplecomprises the steps of: (a) collecting a fecal sample in a transportmedium at normal ambient temperature; (b) dispersing the fecal sample insaid transport medium diluted with a suspension medium; (c) sedimentingcells present in the diluted transport medium of step (b) to isolate thecells from impurities by layering the cell suspension over a medium ofheavier density; then (d) centrifuging the cells in step (c) to form acellular band at a boundary with said heavier medium and within theheavier medium and pellet thereby obtaining a substantially isolatedintact viable exfoliated colonocytes from said fecal sample; and thenreacting said substantially isolated colonocytes with reagents thatselectively bind to leucocytes and inflammatory epithelial cells presentin said substantially isolated colonocytes, wherein the occurrence of apositive reaction of said leucocytes and inflammatory epithelial cellswith said reagents being indicative of the presence of inflammatorybowel disease.
 2. The method of claim 1, wherein said reagents areantibodies against both CD45 and COX-2 antigens.
 3. The method of claim2, wherein the number of leucocytes and inflammatory epithelial cellsreacting to said antibodies are counted, said number being a directmeasure of severity of inflammatory process and progression of thedisease during course of treatment.
 4. A method for diagnosinginflammatory bowel disease, comprising the steps of substantiallyisolating colonocytes from a stool sample by: (a) collecting a fecalsample in a transport medium at normal ambient temperature; (b)dispersing the fecal sample in said transport medium diluted with asuspension medium; (c) sedimenting cells present in the dilutedtransport medium of step (b) to isolate the cells from impurities bylayering the cell suspension over a medium of heavier density; then (d)centrifuging the cells in step (c) to form a cellular band at a boundarywith said heavier medium and within the heavier medium and pellet,thereby obtaining substantially isolated intact viable exfoliatedcolonocytes from said fecal sample; and then reacting said substantiallyisolated colonocytes with antibodies against both CD45 and COX-2antigens wherein the occurrence of cells that bind with said antibodiesbeing indicative of inflammatory bowel disease.
 5. The method of claim4, wherein the number of cells reacting to said antibodies are counted,said number being a direct measure of severity of inflammatory processand progression of the disease during course of treatment.